The present invention relates to a phospholipid composition which is administered by various routes for treating disorders of the central nervous system, particularly those connected with aging of the brain.
Phospholipids are a large class of lipids characterized by a glycerol portion which binds phosphate and fatty acids. The general formula in phospholipids is as follows: ##STR1## wherein X is a particular polar head group characterizing the different phospholipid classes. (See Lehninger, Biochemistry, 2 Ed., p. 288).
It is understood that the role of phospholipids in haematic coagulation is crucial but not yet altogether clear. Indeed, on a molecular level, the interaction of phospholipids during this process with coagulation factors is still to be explained.
By outlining the intrinsic and extrinsic coagulation pathways it is possible to observe that phospholipids play a key role in the coagulation cascade. In fact, phospholipids constitute 30-50% of crude thromboplastin preparations, and their composition varies according to the origin of the preparation. The major part, however, is constituted by phosphatidylcholine (PC) and phosphatidylethanolamine (PE), while negatively charged phospholipids such as phosphatidylserine (PS) and phosphatidylinositol (PI) account for a small part (Liu D. T. H. et al. Thromb. Res. 7 (1975) 213-221). The polar group of the phospholipid plays an essential role in the formation of the complexes in the coagulation pathway (Otnaes A. B. et al Eur. J. Biochem. 27, (1972), 238-243; Nemerson Y. Adv. Exp. Med. Biol. 63, (1975) 245-253).
Regarding the specificity of the polar head, it has been observed that by recombining the tissue factor apoprotein with PE, thromboplastic activity is completely restored (Nemerson Y., supra; Liu D. T. H. et al., supra). PC proves to be less active, while other phospholipid fractions and PE have no activity at all, despite the fact that they bind to the apoprotein fraction. According to Wijngaards et al. (Biochem. Biophys. Acta 488, (1977), 161-171) lipid mixtures with moderate negative charges are necessary for a complete restoration of thromboplastin activity.
Also, the role of externally added phospholipids is not altogether clear. Indeed, there is evidence in the literature that phospholipid preparations from cerebral tissue, prepared by extraction in chloroform and dried in acetone, act in vitro as strong coagulants (Bell, W. M. et al. Nature 174 (1954), 880-881), while phospholipid mixtures extracted from cerebral tissue with chloroform:methanol followed by removal of the nonlipidic parts with saline have proved to be virtually inactive (Folch, H. et al. J. Biol. Chem. 226, (1957), 497-509). It should be noted that in the first case cerebral tissue proteins were present, while in the second case they had been eliminated by the partitioning process.
The composition and purity of the phospholipids used is still the fundamental factor in understanding their exact role, considering also the fact that different activities have been reported for the individual particular classes of phospholipid. It is, however, the correlation between negative charge and coagulant activity which makes phosphatidylserine particularly interesting for these phenomena. The activity of PS is debated with some authors in fact claiming that PS has a catalytic action (Marcus A. J. Adv. Lip. Res. 4 (1966) 1-37), while others report an inhibitory activity of PS (Turner D. L. et al. J. Lip. Res. 5 (1964) 616-623) due to the formation of inactive complexes. Mustard et al. also report (Nature, 196 (1962) 1963-1065) an anticoagulant activity of PS when injected in vivo in dogs in a range of dosages between 30 and 50 mg/kg. In addition, it has been reported that PE has a marked coagulant activity (Rouser et al. Biochem. Biophys. Acta 28 (1958) 71-80; Turner D. L. et al. J. Lip. Res. 4 (1963) 52-56). The activity of a mixture of at least two classes of phospholipid is, however, probably more interesting than that of the single classes.
Some preparations with considerable activity are binary compositions with PE/PS (65/35 w/w), PC/PS (57/43 w/w), PC/PG (48/52 w/w and PG/stearoylamine (85/15 w/w) (Zwaal R. F. A. Biochem. Biophys. Acta 515, (1978), 163-205).
The binary mixture of PS/PC in the presence of calcium seems important in the formation of the coagulation pathway complexes (Subbaiah E. V. et al. Biochem. Biophys. Acta 444, (1976), 131-146). When PS constitutes 25% of the mixture with other phospholipids it proves to be critical in determining, in this range, the maximum biological activity in the conversion from prothrombin and thrombin (M. F. Lecompte et al. J. Electro. Anal. Chem. 104 (1979) 537-541).
It should be observed that this phospholipidic fraction, namely phosphatidylserine, is also interesting for other biological and pharmacological activities.
In fact, a wide range of implications of phosphatidylserine in various biological processes has been reported in the literature. In summary, the biological implications of PS in physiological phenomena are the following:
(a) PS restores ASPase activity associated with isolated neuronal membranes (Wheeler and Whittam (1970), Nature 225, 449-450; Palatini et al (1977) Biochem. Biophys. Acta 466, 1-9).
(b) PS stimulates activity of isolated tyrosine hydroxylase enzyme (Lloyd and Kaufmann (1974) Biochem. Biophys. Res. Comm. 59, 1262-1269; Raese et al. (1976) Biochem. Pharmacol. 25, 2245-2250).
(c) PS liposomes induce cell fusion (Papahadjopoulos et al. (1973) Biochem. Biophys. Acta 323, 23-42).
In addition to the biochemical effects observed, several lines of investigation have shown that cerebral metabolism is influenced by the in vivo administration of PS; the activity of dopaminergic and cholinergic systems is greatly affected, probably by an intervention on presynaptic mechanisms regulating neurotransmitter release. This activity is reflected in measurable effects on several neuroendocrine, electrophysiological and behavioral correlates.
In particular, Bruni et al (Nature 260 (1976) (5549), 331-333) reported a significant biological effect of PS in vivo on cerebral carbohydrate metabolism by measuring an increase of the cerebral glucose haematic ratio. Other phospholipids proved unable to induce this effect.
This cerebral activation after injection in vivo of PS was confirmed by Toffano et al (Life Sciences 23, (1978) 1093-1102), who showed that in cerebral neurotransmitter systems the intravenous (i.v.) injection of PS increased the turnover rate of norepinephrine in the hypothalamus. This hypothalamic effect was accompanied by an increase in the affinity of tyrosine hydroxylase for its synthetic pteridine cofactor (Toffano, Battistin, "Neurochemistry and Clinical Neurology", (1980) 205-214) and by an increase of cAMP.
These effects are correlated with an increase of acetylcholine (ACh) output from the cerebral cortex in urethane-anaesthetised rats (Casamenti et al. J. Neurochem. (32 (1979) 529-533). When ACh levels in rat cerebral cortex were measured, it was found that PS (30 mg/kg i/p. for 10 days) potentiated the effect of the antimuscarinic drug scopolamine (Mantovani et al., Phospholipids in the Nervous System-Vol. 1, (1981) 165-172).
The effect of PS on cerebral neurotransmitter systems is supported by neuroendocrine, electrophysiological and behaviorial correlates. Canonico et al. (Neuroendocrinology 33, (1981) 358-362) described the effects of PS on prolactin secretion in rats. The drug, in acute or repeated administration, reduced plasma prolactin levels during different phases of the circadian rhythm and inhibited the plasma prolactin surge typical of the proestrus afternoon in female rats.
On the other hand, a series of experiments (Mantovani et al., supra; Toffano et al., New Trends in Nutrition Lipid Research, and Cardiovascular Diseases, (1981) 91-99) showed that the typical disruption of EEG pattern induced by the anticholinergic drug scopolamine was efficiently antagonized by previous or simultaneous administration of PS.
Subsequently, Aporti et al. (Res. Comm. Psychol. Psych. Behav. 7 (1982) (1), 131-143) extended the EEG investigations by using computerized analysis of EEG recorded by means of implanted cortical and subcortical electrodes in freely moving rats. Acute administration of PS produced, at a cortical level, a reversal of the pre-drug ratios of the amplitudes in the right and left hemispheres. The effect became most pronounced at the end of 10 days of chronic administration.
This effect is correlated to a typical instinctive explorotory behavior, defined as alternation, and exhibited by rats placed in a T-maze set-up. Also this typical behavior is disrupted by the anticholinergic drug scopolamine. Pepeu et al. (Aging Br. Dementia 13, (1980) 271-274) reported that intraperental (i.p.) administration of PS completely antagonized the disrupting effect of scopolamine on spontaneous alternation in a T-maze.
More recently, Toffano et al. (International Multidisciplinar Seminar: "Cerebral Pathology in Old Age", Second Edition (In press 1983)) reviewed pharmacological data obtained in aged animals, in order to evaluate the protective effects of PS on parameters which undergo changes with age. Injection of PS favorably influenced several biochemical membrane parameters, including stimulation of (Na.sup.+ K.sup.+)-ATPase, and antagonism of age-related alternations of the cholesterol to phospholipid molar ratio. Concomitantly, PS prevented the age-dependent decrease of dopamine and its catabolites in rat striatum and limbic areas.
In addition, a series of studies confirmed a favorable effect of PS on learning and memory processes in aged rats. Positive effects of PS on cognitive functions in aged rats were reported by Drago and Scapagnini, Neurobiology of Aging 2, (1981) 209-213.
In summary, pharmacological data reported in the literature make it evident that administration to man of pharmaceutical compositions containing PS of cerebral origin and with a high degree of purity may represent a valid therapeutic means for treating aging of the brain where a diminishing of dopaminergic control is well known. On the other hand, data reporting the effects of PS on blood coagulation suggest a grave risk of haemorrhage upon administration of PS. This is a particular disadvantageous risk in elderly patients who often already suffer from vascular complications.